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=== E. ChIP-Seq Library Amplification by PCR === * I would initially do a quick qPCR to test the PCR cycle number ::{| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 100ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 15ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''6.5 rxn''' |- | SeqAmp PCR Buffer (2X)||50.00||7.50||48.75 |- | Forward PCR Primer (12.5uM)||2.00||0.30||1.95 |- | Reverse PCR Primer (12.5uM, R3)||2.00||0.30||1.95 |- | 50X SYBR||0.40||0.06||0.39 |- | SeqAmp DNA Polymerase||2.00||0.30||1.95 |- | Total||56.40||8.46|| |} :- Aliquot 8.46ul + 6.6ul of DNA template :- Split 4.5ul to 3X 8-tube strip :- '''ChIP-PCR:''' 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] X16, 18 19 --> 4C hold '''Note:''' I used REalplex (Eppendorf) for amplification<br> :- Load all 4.5ul PCR product in 6% TBE gel. * Note: I did 19 cycles because I wanted to see the background of regular reaction (-BIS) when I ran PCR over 18 cycles. [[File:ZhangLab_2 2014-10-31 18hr 58min_LightningMagprep-DNASMRT-16X.jpg| 400px]] [[File:ZhangLab_2 2014-10-31 18hr 58min_LightningMagprep-DNASMRT-18X.jpg| 405px]] [[File:ZhangLab_2 2014-10-31 18hr 58min_LightningMagprep-DNASMRT-19X.jpg|400px]] * From the gel image above, I can observe amplification in +bis/NTC. The level looked lower than +bis/100pg and +bis/1ng input. at 16 and 18 cycles. For 19 cycles, the amplification was getting to saturated. * No amplification in -bis/NTC was observed at 18 cycles, suggesting no contamination during library prep procedures. * At the same input between +bis/100pg and -bis/100pg, the intensity of PCR product from converted DNA no converted DNA. This should be explained by the contamination in +bis/100pg. * I just got response email from Nathalie, the one who developed the template switching protocol. She suggest to compare the yield after PCR between the reaction with different amount of bis-DNA and bis-NTC by Qubit or Bioanalyzer quantification. * I then amplified the left over reversed transcribed DNA template with indexed primers (just in case, we need to verify by sequencing) using the same condition above at 17 cycles. ::{| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 100ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 85ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''6.5 rxn''' |- | SeqAmp PCR Buffer (2X)||50.00||42.50||276.25 |- | Forward PCR Primer (12.5uM, F2)||2.00||1.70||11.05 |- | Reverse PCR Primer (12.5uM, RXX)||2.00||1.70||0.00 |- | 50X SYBR||0.40||0.34||2.21 |- | SeqAmp DNA Polymerase||2.00||1.70||11.05 |- | Total||56.40||47.94|| |} :- Aliquot 46.24ul to each DNA template tube containing 37.4ul DNA template + 1.7ul of Reverse PCR primer ==== Illumina indexing ==== '''CT:''' ClonTech control fragmented human gDNA :{| {{table}} border= 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Template''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' |- | BIS-CT 1ng||R1 |- | BIS-CT 100pg||R2 |- | BIS-NTC1||R4 |- | BIS-NTC2||R5 |- | CT-100pg||R6 |- | NTC||R7 |} *- Mix well :- Split 42ul to 2X 8-tube strip :- '''ChIP-PCR:''' 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] X17 --> 4C hold :- I did 1 round of AMPure bead purification using 1X AMPure bead. The protocol suggested to use 0.9X to remove all fragment below 250bp. I saw the amplicon size close to 200bp, so I use 1X volume. :- I did not use resuspension buffer provided with the kit, but use EB buffer to elute (25ul). It's quite difficult to to resuspend the beads since the component of PCR mix is quite sticky. In the future , I might try using library resuspension buffer from the kit. :- Measure concentration of each sequencing library by Qubit dsDNA HS assay (2ul for assay) and calculate the yields.
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